Journal: Biomolecules

Article Title: Serum EphA2 as a Promising Biomarker for the Early Detection and Diagnosis of Colorectal Cancer

doi: 10.3390/biom14121504

Figure Lengend Snippet: The analysis of extracellular release, and the intracellular and cell surface expression of EphA2: ( A ) EphA2 in cell culture supernatants was measured by an ELISA at each incubation time. ( B , C ) Western blotting was used to measure and quantify the total EphA2 content of the cells. ( D , E ), The expression of EphA2 on the cell surface was analyzed by flow cytometry using ELISA capture and detection antibodies.

Article Snippet: Serum samples (100 μL) or recombinant human EphA2 protein (R&D Systems, Minneapolis, MN, USA) diluted with Can Get Signal Solution 1 (TOYOBO, Osaka, Japan) to a final dilution of 1:5 were added in duplicate in each well.

Techniques: Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Incubation, Western Blot, Flow Cytometry

Journal: Biomolecules

Article Title: Serum EphA2 as a Promising Biomarker for the Early Detection and Diagnosis of Colorectal Cancer

doi: 10.3390/biom14121504

Figure Lengend Snippet: Soluble EphA2 levels in healthy individuals and colorectal cancer (CRC) patients. ( A ) EphA2 levels in healthy individuals and overall cancer patients (Stages I–IV). ( B ) EphA2 levels in healthy individuals and cancer patients according to stage. Circles represent individual data points, and asterisks indicate statistically significant differences ( p < 0.05).

Article Snippet: Serum samples (100 μL) or recombinant human EphA2 protein (R&D Systems, Minneapolis, MN, USA) diluted with Can Get Signal Solution 1 (TOYOBO, Osaka, Japan) to a final dilution of 1:5 were added in duplicate in each well.

Techniques:

Journal: Biomolecules

Article Title: Serum EphA2 as a Promising Biomarker for the Early Detection and Diagnosis of Colorectal Cancer

doi: 10.3390/biom14121504

Figure Lengend Snippet: The ROC curve analysis of serum EphA2 levels in healthy individuals and colorectal cancer patients (AUC = 0.9, sensitivity = 93.8, specificity = 78.9, cutoff value = 448).

Article Snippet: Serum samples (100 μL) or recombinant human EphA2 protein (R&D Systems, Minneapolis, MN, USA) diluted with Can Get Signal Solution 1 (TOYOBO, Osaka, Japan) to a final dilution of 1:5 were added in duplicate in each well.

Techniques:

Journal: Biomolecules

Article Title: Serum EphA2 as a Promising Biomarker for the Early Detection and Diagnosis of Colorectal Cancer

doi: 10.3390/biom14121504

Figure Lengend Snippet: Serum EphA2 in CRC patients based on clinical characteristics. “N.S.”: Not Significant.

Article Snippet: Serum samples (100 μL) or recombinant human EphA2 protein (R&D Systems, Minneapolis, MN, USA) diluted with Can Get Signal Solution 1 (TOYOBO, Osaka, Japan) to a final dilution of 1:5 were added in duplicate in each well.

Techniques:

Journal: bioRxiv

Article Title: Augmenting CAR NK cell Anti-tumor Activity by Synapse Tuning

doi: 10.1101/2022.10.26.512898

Figure Lengend Snippet: (a) Chimeric antigen receptor design schemes. Antigen recognition domain (anti-EphA2 scFv): green, short hinge domain IgG (SH): grey, transmembrane domain CD28 (CD28TM): salmon, CD28 co-stimulatory domain: purple, CD3ζ activation domain: blue, PDZbm scaffolding anchor domain: red, and mutated PDZbm domain in black (T2A.truncated CD19 tag of the retroviral vector not shown). (b) Fluorescent confocal microscopy of NK cells plated on a poly-L-lysine coated slide with and without recombinant human EphA2 protein incubated for 30 minutes, white bars indicate 10 microns; pZAP70 (green), Lamp1 (magenta), Actin (white). Dashed line in merged images delineate the approximate line scan quantization of the fluorescent signal depicted in the histograms to the right. Quantified single cell fluorescent data is plotted in (c-e). (c) Quantification of synaptic area determined by actin immunolabeling, n=17 and 19, UN; n=19 and 16, CAR Δ; n=76 and 44, CAR; n=12 and 83, CAR.PDZ at 0 and 30 minutes respectively. Two-Way ANOVA was used to determine statistical significance with Two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli to correct for FDR. Statistical difference delineated by q<0.05 *, q<0.01 **, q<0.001 ***; mean±SEM shown. (d) pZAP70 intensity quantification from (c). Arbitrary Units (a.u.), defined by the intensity of the fluorescent signal per unit area of the synapse (actin ring). All cells were normalized to the mean of 0 minutes. Two-Way ANOVA was used to determine statistical significance with Two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli to correct for FDR. Statistical difference delineated by q<0.01 **, q<0.0001 ****; mean±SEM shown. Cells are the same as in (c). (e) Lamp1 intensity quantification. a.u. defined by the intensity of the fluorescent signal per unit area of the synapse (actin ring). All cells were normalized to the mean of no EphA2; mean±SEM shown. Cells are the same as in (c). (f) Confocal images as prepared in (b) incubated for 60minutes with NK cells in various groups quantified in (g,h). White bars indicate 10microns. Immunolabelling of Scribble in red, CD3ε in green, and filamentous actin (F-actin) in white. (g) Scribble polarization and accumulation at the immune synapse (IS). CAR Δ; n=32, CAR; n=23, CAR.PDZmut: n=57, CAR.PDZ n=28 from two independent experiments, One-Way ANOVA was used to determine statistical significance with Two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli to correct for FDR. Statistical difference delineated by q<0.01 **, q<0.001 ***; mean±SEM shown. (h) CD3ε co-localization with Scribble as determined by Pearson Correlation Coefficient. CAR Δ; n=33, CAR; n=48, CAR.PDZmut: n=29, CAR.PDZ n=61, One-Way ANOVA was used to determine statistical significance with Two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli to correct for FDR. Statistical difference delineated by q<0.05 *, q<0.0001 ****; mean±SEM shown.

Article Snippet: Antigen coated coverslips were prepared using N1 coverslips (Fischer Scientific: #12-545-80P), which were coated with 0.5 μg/mL of rhEphA2 (R&D Systems, Minneapolis, MN, USA: #3035-A2-100) or poly-L-lysine (Sigma: #P4707) overnight at 4°C.

Techniques: Activation Assay, Scaffolding, Retroviral, Plasmid Preparation, Confocal Microscopy, Recombinant, Incubation, Immunolabeling